PROJECT SUMMARY TR&D Project 2. The Analysis Stage I: Tools for Analyzing the Composition and Stoichiometry of Macromolecular Assemblies The determination and quantitation of proteins within assemblies remain a significant challenge, as proteins carry much of the burden of the rapid dynamic responses of the cell and are incredibly diverse in their abundance and their physico-chemical properties, making them highly versatile for the dynamic tasks at hand but at the same time difficult to analyze. This TR&D will allow the researcher to quantitatively characterize the constitution of isolated assemblies, and achieve this objective with a fraction of the time, effort, and resources normally required. Thus, we will develop methods to faithfully and fully identify the bone fide components of any target macromolecular complex in the cell, as well as that of vicinal associators, with high accuracy and throughput. We will advance current quantitative approaches for determining complex composition. We will also provide the community with a suite of options for discriminating between specific interactors and contaminants.We will also develop methods to faithfully and fully identify with high accuracy the stoichiometry of any target macromolecular complex in the cell. We will provide this technology to the community in the form of a ?Stoichiometry Package?. This package will incorporate a parallel suite of techniques that give complementary and cross-verifying information, collectively providing accurate, comprehensive sets of stoichiometry data (and dynamic changes) for an isolated assembly. Users can choose any or a combination of approaches, depending on need. We will also gather data that inform on the dynamics of macromolecular complexes, by isolating complexes in defined sequences in space and time; comparisons of how the complexes change with time or different assembly state in terms of composition and stoichiometry will allow reconstruction of these dynamic processes. The major challenge we are addressing here is to achieve these goals with high fidelity in a mode that is readily transportable to any researcher.